ENVIRONMENT AND HEALTH. Evaluation of the Efficacy of Bentonite from the South of Argentina to Ameliorate the Toxic Effects of Aflatoxin in Broilers

ENVIRONMENT AND HEALTH Evaluation of the Efficacy of Bentonite from the South of Argentina to Ameliorate the Toxic Effects of Aflatoxin in Broilers C.A.R. Rosa,* R. Miazzo, C. Magnoli, M. Salvano, S. M. Chiacchiera, S. Ferrero,# M. Saenz, E.C.Q. Carvalho, and A. Dalcero,1 *Departamento de Microbiologia e Imunologia Universidade Federal Rural do Rio de Janeiro-Instituto de Veterinária, Brazil; Departamento de Producción Animal, Facultad de Agronomía y Veterinaria; Departamento de Microbiología e Inmunología; Departamento de Biología Molecular; Departamento de Química y Física; #Departamento de Matemáticas, Facultad de Ciencias Exactas Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Río Cuarto (5800), Córdoba Argentina; and Departamento de Patologia Faculdade de Veterinária Universidade Federal Fluminense, Brazil ABSTRACT In vitro studies indicated that a sodium ameliorated when SB was used in the broiler chick diets. bentonite (SB) from southern Argentina had a high ability to sorb aflatoxin B 1 (AFB 1 ) from aqueous solution. We evaluated this compound for its ability to reduce the effects of total aflatoxins (AF; 5 mg AFB 1 /kg) in the diet of growing broiler chickens from 30 to 52 d of age. The The AF significantly (P < 0.05) decreased feed efficiency. Liver, kidney, and pancreas relative weights increased in chickens fed the diet containing AF alone. Alterations in the levels of serum total protein, albumin (ALB), and globulins (GLOB) were observed for AF diets, and moderate diets were amended with 0.3% Argentinean SB to determine protection was provided by the sorbent. The the effect of this compound during aflatoxicosis. When compared with the controls, BW gains were significantly (P < 0.05) lower for broilers fed diets containing AF alone (1,865 vs. 1,552 g). No differences were found between the BW gains of broiler chickens fed diets without AF (1,785 g) and those of chickens fed AF + SB (1,809 g). These results suggest that effects of AF treatment were ALB:GLOB ratio decreased in both groups of birds fed with the AF-contaminated diet, and we observed a moderate increase in this ratio by 0.3% addition of SB. The histopathological findings in liver sections of broiler fed diets with AF + SB indicated a nonprotective effect of this adsorbent, because a moderate hepatic steatosis was observed. (Key words: aflatoxin, adsorbent, bentonite, detoxification, broilers) 2001 Poultry Science 80:139 144 INTRODUCTION Fungal contamination of agricultural products is often unavoidable and of worldwide concern, because the products often contain toxic metabolites. Mycotoxins cause a wide variety of adverse clinical signs, depending on the nature and concentration of toxins in the diets, on the animal species, on their age, and on nutritional and health status at the time of exposure to contaminated feed (Dmello and Macdonald, 1997). Definitive ways for complete detoxification of mycotoxin-contaminated food and feed do not exist; therefore, new methods to eliminate mycotoxicosis are sought (Doyle et al. 1982; Bauer, 1994). Aflatoxins (AF), a group of mycotoxins that can be present as contaminants in a large number of food and feedstuffs, are the most dangerous of these fungal second- ary metabolites. Aflatoxins are highly toxic, mutagenic, teratogenic, and carcinogenic compounds (Miller, 1995). Occurrence of AF and other mycotoxins in poultry and animal feedstuffs is quite common in many countries, including Argentina (Dalcero et al., 1997; 1998). In previous work (Magnoli et al., 1998), a high prevalence and an elevated percentage of species of section Flavi (Aspergillus flavus var. flavus and Aspergillus flavus ssp. parasiticus) were toxigenic. In poultry aflatoxicosis, inappetency, faintness, weakness, and frequently ataxia can be observed. Pathological investigation may reveal a swollen and yellowish fatty liver with hemorrhages on its surface and swelling of kidneys. Histopathological changes reveal disorganization of the hepatic structure (dystrophy) and a severe necrosis of parenchymal cells (fatty necrosis infiltration) that can be accompanied by a proliferation of bile vessels Received for publication November 2, 1999. Accepted for publication July 26, 2000. 1 To whom correspondence should be addressed: adalcero@exa. unrc.edu.ar. Abbreviation Key: AFB 1 = aflatoxin B 1 ;ALB= albumin; AF = aflatoxin; GLOB = globulin; SB = sodium bentonite. 139

140 ROSA ET AL. and fibrosis (Bata et al., 1996). Therefore, a practical and effective method for detoxification of AF-containing feedstuffs is an urgent requirement. Although large-scale and cost-effective methods for complete detoxification of mycotoxin-contaminated feedstuffs are currently not available, one of the more encouraging approaches is the addition of nonnutritive sorptive materials to feedstuffs with the consequent reduction of the gastrointestinal absorption of these fungal metabolites (Piva et al., 1995). The efficacy of the adsorbents has been shown to be true mainly against polar mycotoxins, particularly AF (Ramos and Hernandez, 1997). Layered aluminosilicates such as bentonites are already commercially used for the detoxification of AF-contaminated feedstuffs (Kubena et al., 1990b, 1998; Abo-Norag et al., 1995; Ramos and Hernández, 1997). However, adsorption ability of these clays may vary from one geological deposit to another. At gizzard ph, a total delamination of some aluminosilicates can occur. Because the binding spectra of these substances are quite broad and nonspecific, nutritional components such as vitamins may also be removed (Ramos et al., 1996). The objective of this experiment was to evaluate the efficacy of a natural sodium bentonite (SB) from southern Argentina to reduce bioavailability and to diminish the toxic effects of AF in broiler chickens. Chemicals MATERIALS AND METHODS We purchased aflatoxin B 1 (AFB 1 ), standard purity >99%, 2 and the purity was assayed by HPLC. Commercial SB del Lago (SB) was obtained from a mine situated in Cinco Saltos, Province of Río Negro, Argentina. The chemical composition of the SB was 54.91% SiO 2, 21.41% Al 2 O 3, traces of Fe 2 O 3, 0.01% MnO, 0.1% TiO 2, traces of CaO, 2.81% of MgO, 1.70% of Na 2 O, 0.16% K 2 O, traces of SO 3, 0.05% P 2 O 5, and 5.59% H 2 O. The mean particle size was 53 µ (99.5%). A 6% SB aqueous solution had ph of 8.5, and the swelling capacity after 24 h was 16 cm 3 /g. In Vitro Studies Adsorption Isotherm. A stock of methanolic AFB 1 solution containing 220 µg AFB 1 /ml was prepared. The working solutions were made from the stock by dilution with buffered water at ph 2 (0.05 M KCl 0.013 M HCl) to a final concentration of 8, 21, 28, and 51 µg ml 1. Samples of sorbent (11 mg) were weighed into clean glass tubes (two replicates), and a total of 10.0 ml of each working solution was added at timed intervals, so that the total incubation period was equal to 1 h for each tube. Controls of AFB 1 without sorbent and blanks prepared with buffered water at ph 2 and sorbent were also in- 2 Sigma Chemical Company, St. Louis, MO 63103. cluded. All tubes were shaken for 1 h at 39.5 ± 1 C. After incubation, the solutions were centrifuged for 5 min at 16,060 g, and the supernatant was decanted carefully into a clean tube. The AFB 1 concentration in each supernatant was determined by UV-visible spectrometry and HPLC. Percentage sorption of AFB 1 was estimated from the amount of unbound (free) AFB 1 remaining in the supernatant after incubation. Effect of Salt Concentration. The working solutions were made from the AFB 1 methanolic stock solution (at 220 µg -1 ml) by dilution with 1.5 M NaCl buffered ph 2 water solution to a final concentration of 51 µg -1 ml. The adsorption ability was determined as described above. Stability of Bentonite-AFB 1 Complex. Test samples (0.5 g) of dry bentonite, containing 2.5 µg of adsorbed AFB 1, were added to test tubes and mixed thoroughly with 5 ml of each of the following solvents: water, methanol, and chloroform. Water was tested at ph 2, 7, and 10 and temperatures of 25 and 37 C. The bentonite was pelleted by centrifugation at 16,060 g for 5 min, and 1- ml aliquots of each supernatant were removed. The total percentage of desorption was determined for each sample. In Vivo Studies AF Production and Analysis. Aflatoxins were produced via fermentation of milled corn by Aspergillus parasiticus NRRL 3000. The sterile substrate, placed in Erlenmeyer flasks, was inoculated with 2 ml of the mold aqueous suspension containing 10 6 spores-1 ml. Cultures were allowed to grow for 7 d at 25 C in darkness. On the seventh day, Erlenmeyer flasks were autoclaved, and culture material was dried at 40 C in a forced-air oven for 48 h. The AF content was measured by HPLC according to Trucksess et al. (1994) and AOAC (1995). The AF content in the milled corn was 84% AFB 1,8%AFG 1,6%AFB 2, and 2% AFG 2. The milled corn was incorporated into the basal diet to provide the desired level of 5 mg AFB 1 /kg of diet. Experimental Design. Thirty-day-old Arbor-Acres male chickens (five replicates per treatment with five chickens per replicate and four treatments) obtained from a commercial hatchery were fed for 22 d under standard management conditions with commercial feedstuffs and water available ad libitum. Chickens were fed a corn basal diet (commercial type with added antibiotics, coccidiostatics, or growth promoters) containing approximately 19% protein, 3.5% fiber, 5.1% fat, and 3,100 Kcal ME/kg to meet or exceed the levels of critical nutrients recommended by the NRC (1984). During the experimental period, the control diet was analyzed for mycotoxins: zearalenone, deoxynivalenol, and fumonisins. Levels found were below the detection limits of previously described techniques (Dalcero et al., 1997). Levels of naturally occurring AFB 1 ranged from 0 to 11 µg/kg. Experimental diets for each treatment were as follows: 1) control, feedstuffs without addition of bentonite and AFB 1 ; 2) 0.3%

SB; 3) 5 mg AFB 1 /kg feed; and 4) 5 mg AFB 1 /kg feed plus 0.3% SB. Chickens were monitored daily for signs of morbidity and mortality. The effect of SB on aflatoxicosis was determined by measuring BW; feed:gain ratio; and total protein, albumin (ALB), and globulin (GLOB) concentrations of serum of broilers in each group. These concentrations were determined with a clinical chemistry analyzer 3 according to the manufacturer s recommended procedure. The birds were killed by cervical dislocation and were necropsied and examined for gross lesions. Histopathology EFFICACY OF BENTONITE 141 Livers were excised, weighed, and fixed in 10% neutral buffered formalin. Fixed tissues were trimmed, embedded in paraffin, and stained with hematoxylin and eosin for histopathological examination. Liver samples were also cut on a freezing microtome and stained with Sudan III. Liver sections of all birds were microscopically examined. Statistical Analysis Data were evaluated with ANOVA for a complete randomized design, using the general linear models procedure of SAS software (SAS Institute, 1985). When the ANOVA showed significance, Tukey s significant-difference test was applied. RESULTS To test the in vitro binding ability of SB to sorb AFB 1, isotherm adsorption at ph 2 and 39.5 C was carried out (Figure 1). From the plot, the maximum adsorption capacity of SB for AFB 1 was estimated to be 45 µg/mg SB. To test the in vitro effect of high salt concentrations on the capacity of SB to bind AFB 1, we kept other experimental conditions constant but in presence of 1.5 M NaCl. The presence of the salt did not affect the binding capacity of SB. The complexed SB-AFB 1 was stable to solvent extraction; less than 10% of the AFB 1 sorbed by SB was extracted, suggesting a strong bond formation (chemisorption). These results suggest that SB would be a good candidate for in vivo testing in birds. Addition of 0.3% SB was estimated to be at least 30 times higher than amount needed to bind 5 mg AFB 1 / kg feed in an in vivo assay. The effects of AF and SB diets on broiler performance are presented in Table 1. Our results showed that 0.3% SB in the diets significantly diminished the inhibitory effects of feeding 5 mg AFB 1 /kg diet. Mortality did not occur. Compared with the controls (basal diet and basal diet + adsorbent), a significant decrease in BW gains (17%) was observed in birds fed AF alone (P < 0.05), but no 3 Metrolab 2.100. FIGURE 1. Isotherm plot for aflatoxin B 1 adsorption to sodium bentonite (SB) at ph 2 and 39.5 ± 0.5 C. differences in BW gains were noted in diets without AF. The feed:gain ratio (kg feed/kg gain) was significantly (P < 0.05) increased by AF intake. The other treatments did not show significant differences. Reduced levels of serum total protein, ALB, and GLOB are indicators of aflatoxicosis (Table 1). Levels of these biochemical parameters decreased (P < 0.05) in both treatments with 5 mg AFB 1 /kg diet, but the decrease was not as great when 0.3% SB was added to the AF-contaminated diet. The ALB:GLOB ratio also decreased in both groups of birds fed AF-contaminated diets. Addition of 0.3% SB caused a moderate increase in this ratio. Data presented in Table 2 show the effects of dietary treatments on relative organ weights (g/100 g BW). The diet containing 5 mg AFB 1 /kg without bentonite added increased relative liver, kidney, and spleen weights. However, addition of 0.3% SB to the AF-contaminated diet prevented an increase in weight of these organs. Livers of broilers fed diets containing AF were friable and pale in appearance, whereas livers of broilers that consumed other diets were normal in apearance. Representative livers of broilers fed diets containing AF, SB, and controls at 21 d of treatment are shown in Figure 2. When AF was given to birds, histopathologic changes included moderate to severe diffuse hepatic vacuolization in the hematoxylin-eosin stain preparation (Figure 3a). The microvacuolization was coalescent, and its content was strongly sudanophilic, characterized by severe and diffuse hepatic steastosis (Figure 3b). Addition of 0.3% SB to the diet containing 5 mg AFB 1 / kg caused liver changes that included moderate, diffuse, and sudanophilic citoplasmatic microvacuolization with moderate and diffuse hepatic steatosis (Figure 4a). Livers of the group fed the control diet or the control plus SB diet showed cloudy, discreet, and diffuse cytoplasmatic swelling (Figure 4b). The sudanophilic stain was negative.

142 ROSA ET AL. TABLE 1. Effects of sodium bentonite on BW gain 1, feed:gain ratio, 2 and biochemical indicators 1 of broiler chicks fed diets containing 5 mg aflatoxin B 1 (AFB 1 )/kg feed Treatment BW gain Biochemical indicators Changes Serum Serum Serum Ratio of AFB 1 SB 3 30 to 52 from the Feed:gain total albumin globulins serum 5 mg/kg 0.3% d old control ratio protein (ALB) (GLOB) ALB/GLOB (g) (%) (kg/kg) (g/100 ml) 1,865 a ± 31 0 2.32 a ± 0.03 3.83 a ± 0.11 1.73 a ± 0.05 2.10 ± 0.05 0.82 + 1,785 a ± 27 4 2.30 a ± 0.02 3.59 a ± 0.10 1.69 a ± 0.02 1.90 ± 0.04 0.89 + 1,552 b ± 48 17 2.51 b ± 0.03 1.69 b ± 0.25 0.52 b ± 0.08 1.17 ± 0.07 0.44 + + 1,809 a ± 28 3 2.38 a ± 0.04 2.54 a ± 0.24 1.00 a ± 0.07 1.54 ± 0.04 0.65 a,b Values within columns with no common superscripts are significantly different (P < 0.05) according to the Tukey test. 1 Results are reported as the mean ± SD for 25 animals. 2 Results are reported as the mean ± SD for five lots. 3 Sodium bentonite. DISCUSSION Aflatoxins are very important to the poultry industry because of their toxicity (Huff et al., 1992) and occurrence in feedstuffs (Kubena et al., 1993). In vitro results suggested that SB was a strong binder for AFB 1, and they were consistent with those of Veldman et al. (1992), who reported the formation of an inert and stable complex between AFB 1 and bentonite, capable of preventing AFB 1 absorption in the intestine. In this study, experimental aflatoxicosis was induced in 30-d-old broilers by feeding them with 5 mg total AF/ kg diet for 3 wk. In vivo studies demonstrated the known toxicity of AF to the broiler chickens and the ability of SB to ameliorate the toxic effects. Our results agree with those reported by Kubena et al. (1990a, 1993) as regards BW gains in broilers. No significant differences were found between chickens fed the control diet and chickens fed the diets containing SB alone, indicating that the adsorbent was inert and nontoxic. Aflatoxin has been shown to cause inhibition of protein synthesis (Tung et al., 1975; Yu, 1977). As a result, hypoproteinemia is a common effect of aflatoxicosis (Huff et al., 1986). Kubena et al. (1990a,b) reported that decreased serum total protein and ALB in broilers as a result of aflatoxicosis were not alleviated by hydrated sodium cal- cium aluminosilicate. Kececi et al. (1998) have shown that some serum biochemical changes could be ameliorated by bentonite administration to the diet at doses of 5 mg/ kg in broiler chickens given 2.5 mg AF/kg diet. However, in our case, the biochemical parameters for broilers fed diets containing SB + AF did not completely return to normal values, showing an inhibition of the protein synthesis. These results agree with the findings reported by Tung et al. (1975), who observed a decrease in these parameters and an inhibition in protein synthesis during the aflatoxicosis in poultry. The histopathological findings in liver sections of broilers fed diets with AF + SB indicated a nonprotective effect of the adsorbent, because a moderate hepatic steatosis was observed. When these data are compared to preliminary results for which the sorbent used was a synthetic TABLE 2. Effect of sodium bentonite (SB) on relative organ weights of broiler chicks fed diets containing 5 mg aflatoxin B 1 (AFB 1 )/kg feed 1 Treatment AFB 1 SB 5 mg/kg Feed 0.3% Liver Kidney Spleen (g/100 g BW) 2.84 a ± 0.05 0.54 a ± 0.05 0.13 a ± 0.03 + 2.65 b ± 0.03 0.48 a ± 0.04 0.10 a ± 0.02 + 3.22 b ± 0.05 0.63 b ± 0.05 0.18 b ± 0.03 + + 2.63 b ± 0.02 0.46 a ± 0.02 0.10 a ± 0.04 a,b Values within columns with no common superscripts are significantly different (P < 0.05) according to the Tukey test. 1 Twenty-one dayas of treatment. The results are reported as the mean ± SD for 25 animals. FIGURE 2. Representative livers from broiler chickens (29 d old) fed the control diet (a), 0.3% sodium bentonite (SB) additive (b), 5 mg aflatoxin B 1 (AFB 1 )/kg feed (c), and 5 mg AFB 1 /kg feed plus 0.3% SB (d).

143 EFFICACY OF BENTONITE FIGURE 3. Photomicrographs of hematoxylin and eosin stained liver sections of a control chicken (29 d old) that shows discrete and diffuse cloudy cytoplasmatic swelling (A) and sections of liver of a broiler fed 5 mg aflatoxin B1/kg feed that reveled a severe and diffuse hepatic vacuolization (B). Bar equals 10 µm. sodium zeolite, under the same experimental conditions, no hepatic histopathological changes were observed (Miazzo et al., 2000). Further in vivo investigations are required to confirm the ability of these agents to sequester the most important mycotoxins, either singly or in combination, to establish the amount to be added to feeds and to determine any long-term effects they may have on gastrointestinal absorption of essential nutrients. We agree with Dwyer et al. (1997), who suggested that in vitro data predicting the ability of inorganic adsorbents to protect chickens from the adverse effects of mycotoxins should be viewed with caution and should be confirmed in vivo, paying particular attention to the potential for nutrient interactions. The results of the in vitro studies have shown that the SB from southern Argentina had a high ability to sorb AFB1 from aqueous solution. The data from the present study suggested that the formation of the SB-AFB1 complex should decrease the bioavailability at AFB1. However, addition of this compound to feedstuffs contaminated with AF only had a moderate protective effect against the development of aflatoxicosis in these broiler chickens. The reduction in broiler BW gains and the poor efficiency of feed utilization caused by 5 mg AF/kg diet were ameliorated by addition of 0.3% SB to the AF-contaminated diet. Although an improvement in BW gains was observed in broilers fed the AF + SB diet, the biochemical parameters and the histopathological findings were not normal and revealed significant hepatic changes. The results of this study are important because SB could be incorporated into chicken diets economically as it can be obtained from mines of southern Argentina. The results of in vitro studies suggested that SB was a good candidate to ameliorate the toxic effects of AFB1. Body weight gain and feed:gain data agree with the results of the in vitro studies, because SB fed chickens had values similar to those of control chickens. However, the biochemical indicators and histopathological findings showed that the amelioration of AF-toxic effects was not as great as might have been predicted. Similar studies carried out with a synthetic sodium zeolite seem to give better results compared with SB sorbent, not only because of the BW gains but also the histopathological studies (Miazzo et al., 2000). However, it should be pointed out that the age at which the birds were exposed to contaminated feed were different in the two experiments. Taking into account that the clinical signs for aflatoxicosis depend on bird age and nutritional or health status at the time of exposure to contaminated feed, we suggest that further in vivo experiments comparing both adsorbents should be conducted. These are the first data reported from Argentina on dietary addition of nonnutritive, natural, inorganic compounds that could bind AF in the gastrointestinal tract to prevent their absorption. ACKNOWLEDGMENTS The authors are grateful to the SECyT (Secretarı a de Ciencia y Te cnica Universidad Nacional de Rı o Cuarto) Res. No. 235/97; CONICOR (Consejo de Investigaciones Cientı ficas y Tecnolo gicas de la Provincia de Co rdoba) Res No. 1416/97, CONICET (Consejo Nacional de Investigaciones Cientı ficas y Te cnicas) PEI Res. No. 0457/97, which supported this study through grants. C.M. thanks CONICOR for financial support. S.M.C. held a research position at CONICET. Castiglioni y Acimientos, Pesycia. S.A.F.I.A.M.I. for giving us bentonite. FIGURE 4. Photomicrographs of Sudan III-stained liver sections of a chicken (29 d old) fed 5 mg aflatoxin B1 (AFB1)/kg feed; the microvacuolization was coalescent and strongly sudanophilic (severe hepatic steatosis) (A). Liver of a chicken fed 0.3% sodium bentonite plus 5 mg AFB1/kg feed showing moderate, diffuse, and sudanophilic cytoplasmatic microvacuolization (moderate hepatic steatosis) (B). Bar equals to 10 µm. REFERENCES Abo Norag, M., T. S. Edrington, L. F. Kubena, and R. B. Harvey, 1995. Influence of a hydrated sodium calcium aluminosillicate and virginamycin on aflatoxicosis in broiler chicks. Poultry Sci. 74:626 632.

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